Citrate overall reaction

Substrates can affect the conformation of the other active sites. So can other molecules. Effector molecules other than the substrate can bind to specific effector sites (different from the substrate-binding site) and shift the original T-R equilibrium (see Fig. 8-9). An effector that binds preferentially to the T state decreases the already low concentration of the R state and makes it even more difficult for the substrate to bind. These effectors decrease the velocity of the overall reaction and are referred to as allosteric inhibitors. An example is the effect of ATP or citrate on the activity of phosphofructokinase. Effectors that bind specif-... 

The first step has a AG0 of —0.05 kcal/mole, which is close to zero it does not occur to any great extent unless the concentrations of acetyl-coenzyme A (acetyl-CoA) and oxaloacetate are greater than the concentration of citryl-CoA. The second step, however, has a highly favorable AG0 of — 8.4 kcal/mole. When the two steps are combined, AG0 for the overall reaction is about —8.3 kcal/mole, and the equilibrium constant lies far in the forward direction. These two reactions are catalyzed by the enzyme citrate synthase, by a mechanism that ensures that they always occur together. 

With the stereochemistry of the citrate lyase reaction determined, that of the Si citrate synthetase (the common enzyme) was established as shown in Fig. 70. Condensation of (J )-acetic-d, t acid (configuration known by synthesis) with oxalo-acetate gives what turns out to be mainly (2S,3/ )-citric-2-d,2-/ acid (112).41 When this acid is then cleaved with citrate lyase, the major product is (/ )-acetic-d, t acid, as established by the malate synthetase/fumarase diagnosis. It follows that both the Si-citrate synthetase and citrate lyase reactions must involve the same stereochemical course. Since that of the lyase reaction is inversion (vide supra), that of the Si synthetase reaction must be inversion also. And since the overall stereochemical result shown in Fig. 70 is not dependent on the magnitude of the... 

This reaction, which is an aldol condensation followed by a hydrolysis, is catalyzed by citrate synthase. Oxaloacetate first condenses with acetyl CoA to form citryl CoA, which is then hydrolyzed to citrate and CoA. The hydrolysis of citryl CoA, a high-energy thioester intermediate, drives the overall reaction far in the direction of the synthesis of citrate. In essence, the hydrolysis of the thioester powers the synthesis of a new molecule from two precursors. Because this reaction initiates the cycle, it is very important that side reactions be minimized. Let us briefly consider the how citrate synthase prevents wasteful processes such as the hydrolysis of acetyl CoA. 

The starting point of lipid anabolism is acetyl-GoA. The anabolic reactions of lipid metabolism, like those of carbohydrate metabolism, take place in the cytosol these reactions are catalyzed by soluble enzymes that are not bound to membranes. Acetyl-GoA is mainly produced in mitochondria, whether from pyruvate or from the breakdown of fatty acids. An indirect transfer mechanism exists for transfer of acetyl-CoA in which citrate is transferred to the cytosol (Figure 19.13). Citrate reacts with GoA-SH to produce citryl-CoA, which is then cleaved to yield oxaloacetate and acetyl-GoA. The enzyme that catalyzes this reaction requires ATP and is called ATP-citrate lyase. The overall reaction is... 

As pyruvic acid decarboxylation constitutes the link between glycolysis and the Krebs cycle, a-ketoglutaric decarboxylation divides the reactions involving 6-carbon acids (citrate, isocitrate, and oxalosuccinate) and those involving 4-carbon acids (succinate, fumarate, and malate). The analogy between the two reactions is not restricted to their role in intermediate metabolism, but extends also to the mechanism of action of the two multiple-enzyme systems. In a-ketoglutaric decarboxylation, the overall reaction leads to the formation of CO2 and succinate. CoA, NAD, thiamine, lipoic acid, and magnesium are requirements for this multiple-enzyme system activity. 

The overall reaction of the citrate cycle, which is shown in Figure 17.8, is ... 

Citrate is then reversibly isomerized to isocitrate, via a successive dehydration to cis-aconitate and rehydration to isocitrate. The overall reaction is catalysed by aconitase, and Fe and a sulphydryl group are required components. At equilibrium, 90% citrate, 4% cis-aconitate and 6% isocitrate are present... 

Isocitrate dehydrogenase catalyses the synthesis of oxoglutarate from iso-citrate through an oxidation-decarboxylation process. Two isocitrate dehydrogenases are present in the cell. The overall reaction is identical for both enzymes ... 

Polycarboxylic acid synthases. Several enzymes, including citrate synthase, the key enzyme which catalyzes the first step of the citric acid cycle, promote condensations of acetyl-CoA with ketones (Eq. 13-38). An a-oxo acid is most often the second substrate, and a thioester intermediate (Eq. 13-38) undergoes hydrolysis to release coenzyme A.199 Because the substrate acetyl-CoA is a thioester, the reaction is often described as a Claisen condensation. The same enzyme that catalyzes the condensation of acetyl-CoA with a ketone also catalyzes the second step, the hydrolysis of the CoA thioester. These polycarboxylic acid synthases are important in biosynthesis. They carry out the initial steps in a general chain elongation process (Fig. 17-18). While one function of the thioester group in acetyl-CoA is to activate the methyl hydrogens toward the aldol condensation, the subsequent hydrolysis of the thioester linkage provides for overall irreversibility and "drives" the synthetic reaction. 

The transformation of pyruvate to carbon dioxide is achieved by the several steps in a cyclical series of reactions known as the tricarboxylic acid (TCA) cycle. The name of the cycle comes from the first step where acetyl-CoA is condensed with oxaloacetic acid to form citric acid, a tricarboxylic acid. Once citrate is formed the material is converted back to oxaloacetate through a series of 10 reactions, as illustrated in Fig. 5.22, with the net production of 2 molecules of carbon dioxide and reducing equivalents in the form of 4 molecules of NADH + H and 1 molecule of FADH2, together with 1 mole of ATP. The overall stoichiometry of the TCA cycle from pyruvate is ... 

The overall consumption of one molecule of acetyl-CoA in the citric acid cycle is an exergonic process AG° = —60 kJ mol-1. All but two of the individual reactions are exergonic. Step 2 (citrate— isocitrate) and step 8 (malate —>oxaloacetate) are endergonic (Fig. 12-3). 

As an example to illustrate analysis of kinetic data to characterize the mechanism of a real enzyme, here we apply the general compulsory-order ternary mechanism introduced above to citrate synthase to determine kinetic parameters for several isoforms of this enzyme and to elucidate the mechanisms behind inhibition by products and other species not part of the overall chemical reaction. 

Note that because all catalysts (oxaloacetate, enzymes etc.) must be regenerated in looking at the overall operation of the cycle, only the acetyl group of acetyl-CoA can be oxidized completely. Some intermediates, such as citrate, can be partially oxidized, but Kreb s cycle intermediate catabolism requires leaving the cycle at oxaloacetate and then returning as acetyl-CoA. It requires leaving the mitochondria for some reactions, and since the extremely low concentrations of oxaloacetate don t allow its efficient transport across the mitochondrial membrane (the Km of the carrier is much higher than [oxaloacetate]), malate is the species which actually leaves the mitochondria. 

We can see that the overall conversion of fumarate to citrate is favorable iri spite of reaction 2 with its low Aiq. This problem and those preceding it illustrate some general rules and principles summarized below. 

Regeneration. The products formed by treating sulfur dioxide solutions with hydrogen sulfide depend in part on the pH. When hydrogen sulfide is added to a solution of sulfur dioxide in water, a complex mixture is formed that includes polythionic acids, thiosulfuric acid, and colloidal sulfur (in contrast to the crystalline sulfur obtained in the citrate process). This is known as Wackenroder s solution and has been extensively studied. The composition varies with the conditions used. With excess hydrogen sulfide the final product is ultimately approximately 100% sulfur (6). While the overall stoichiometry of the reaction is the same as the gas phase Claus reaction, the chemistry is more complex. 

The overall standard free-energy value for the conversion of citrate to isocitrate is the sum of the two values for the individual reactions. 

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